The present invention relates to methods for the identification of anti-HIV miRNAs and anti-HIV pharmaceutical compounds using high-throughput screening methods, comprising: transfecting reporter cells with a panel of miRNAs, infecting the reporter cells with HIV, screening the cells to identify miRNAs that modulate HIV infection and identifying the specific pathways, nucleic acids and/or polypeptides that are targeted by the miRNAs. The invention further provides for the identification and screening of anti-HIV pharmaceutical compounds having known activity against the specific pathways, nucleic acids and/or polypeptides that are targeted by the miRNAs for efficacy in the treatment of HIV. The invention also provides for the use of miRNA mimics, miRNA inhibitors and pharmaceutical compounds in the treatment and/or prevention of HIV infection.
Humans display a remarkably diverse susceptibility to infection, the foundation of which lies in our genetic variation and ability to respond to selective pressures applied by various infectious agents. The evolution of our complex and multi-player immune system underlines the dominance of the human host following a microbial infection. However, given the nature of obligate intracellular pathogens, their complete reliance on host gene expression machinery has led to the evolution of complex interplays between the two, such that pathogens actively and strategically manoeuvre their way through the host terrain. Our traditional view of this terrain as being comprised of protein-coding genes, translation intermediates (mRNAs) and protein counterparts is far too simplistic, particularly in the context of infection. The discovery of the RNA interference (RNAi) pathway, for which the 2006 Nobel Prize was awarded, has greatly enhanced our understanding of the host terrain. Small noncoding RNAs (ncRNAs) termed microRNAs (miRNAs) were shown to be key regulators of gene expression that function within the RNAi pathway to post-transcriptionally modulate mRNA stability and subsequent translation (Fire et al., (1998)). Indeed, it is now understood that miRNAs are able to rapidly, and with exquisite specificity, modulate gene expression in response to numerous environmental cues in a highly coordinated, complex and tissue-specific manner. Given the reliance of intracellular pathogens on host gene expression machinery, the RNAi pathway, and specifically miRNAs, are now understood to lie at the nexus of the host-pathogen interplay.
Only 2% of the metazoan genome encodes protein, yet more than 50% is transcribed and we have little knowledge regarding these transcripts that function in the absence of protein production. In fact, stable ncRNA transcripts have been referred to as ‘dark matter’ within the cellular environment (Yamada et al., (2003)). Despite improvements in the human draft genome sequence, ncRNAs remain difficult to define and thus quantify (Ponting et al., (2010)). However, numerous evolutionary studies have revealed that ncRNAs are estimated to be expressed at 4-fold excess compared to their protein-coding counterparts and are highly conserved across eukaryotic genomes (Ponting et al., (2010)). Currently, ncRNAs have been classified by size into long ncRNAs (IncRNAs; >200 bp) or small ncRNAs (<200 bp) and comprise 26 or more functional categories reflecting something of the diversity of ncRNA function (Ponting et al., (2010)). In the case of humans, IncRNAs comprise about 48% of the genome (Lander et al., (2001)) while small ncRNAs including miRNAs, constitute a small but significant portion of the genome. While new ncRNAs are rapidly being uncovered, functional data remains sparse particularly at the host-pathogen interface. Endogenous miRNAs are usually transcribed from RNA polymerase II promoters (Lee et al., (2004)) as primary miRNA transcripts (pri-miRNAs) several kb in length, containing various stem-loop structures and ssRNA flanking segments. Pri-miRNAs are processed in two compartmentalized steps via the actions of distinct protein complexes (Lee et al., (2002)) to form mature miRNAs that regulate post-transcriptional protein synthesis by base pairing to cognate mRNAs. Depending on the degree of complementarity between the mature miRNA and its target, multiple mRNA silencing modes can occur (Azuma-Mukai et al., (2008)). The initial bases from positions 2 to 7 of the mature miRNA are termed the ‘seed’ sequence and they provide most of the pairing specificity. In some cases, complete pairing between the seed region and target mRNA is sufficient to mediate cleavage of the cognate mRNA (Liu et al., (2004); Yekta (2004)). More typically for mammalian and viral mRNA targets however, cleavage activity is severely impaired by mismatched pairing in the seed and other regions and translational inhibition occurs (Martinez and Tuschl (2004)). Intriguingly, since the complementary length of seed sequence required for miRNAs to target cognate mRNAs is short, each miRNA can target and modulate hundreds of transcripts. Indeed, current estimates predict that thousands of human transcripts are regulated by miRNAs (Farh et al., (2005); Lim et al., (2005)). Furthermore, a single miRNA can regulate multiple mRNA molecules that can in turn also be acted upon by numerous distinct miRNAs (Barbato et al., (2009)). Importantly, most miRNAs decrease target protein levels by less than 2-fold (Baek et al., (2008)), but this non-linear tuning mechanism can still exert a large physiological effect (Ebert and Sharp (2012)). Thus, the endogenous miRNA pathway represents a highly efficient system to simultaneously fine-tune the expression of numerous genes as well as modulate specific functional pathways.
Considering human cells encode >1000 miRNA species, many of which function in innate immunity, it is unsurprising that pathogens (and viruses in particular) have evolved mechanisms to subvert these cellular components (Cullen (2013)). The particular mechanisms by which viruses manipulate the host immune system are as varied as the viruses themselves but if one focuses on viral interactions with cellular microRNA machinery, the options are surprisingly minimized and constrained to a fairly limited number of human viruses. Furthermore, as miRNAs are expressed in a very tissue specific manner and can vary depending on the cell cycle stage, the interactions between host miRNAs and pathogens is clearly complex. The ability of a host organism to mount an innate immune response after pathogen infection is critical for survival and many cellular mRNAs that control host defences are regulated by miRNAs. The promiscuity of miRNAs in regulating their mRNA targets coupled with their importance in posttranscriptional regulation of host gene expression make unraveling the role of miRNAs at the host-pathogen interface extremely challenging. Resolving these interactions requires identification of the specific pathogen-encoded stimuli that induce changes in the host miRNome following infection, assessment of which transcripts are targeted by miRNAs as well as which miRNAs are responsible, quantification of the miRNA-induced changes to the infection transcriptome, analysis of downstream effects on related protein outputs, and validation of each step to ensure a robust understanding of such a complex network of interactions.
To identify host miRNAs required by the Human Immunodeficiency Virus (HIV-1) during infection, we conducted several genome-wide miRNA-based screens. We identified numerous host miRNAs that inhibit HIV-1 replication or enhance activation of the HIV long terminal repeat (LTR) promoter either when the specific miRNA is over-expressed (thus boosting expression levels of its endogenous miRNA sequence) or suppressed (thus ‘sponging out’ or quenching its endogenous miRNA sequence).
Based on extensive literature searches, the human mRNA targets of each of the HIV inhibitory miRNAs were identified. Intriguingly, many of these mRNA targets code for host proteins that play a role in the cellular response to DNA damage, repair and apoptosis. Given that these cellular pathways are central to the development and control of many cancers, we conducted additional validation experiments which have highlighted the close relationship between early HIV infection and abnormal cell survival phenotypes which are related to the DNA damage pathway and which are characteristic of many cancers. We thus sought to utilise compound libraries comprised of oncogenic and kinase-specific inhibitors, and tested for their ability to inhibit HIV infection. Several of the cancer-specific therapeutics inhibited HIV replication at targets and pathways analogous to those identified in the miRNA screens. Taken together our results have revealed novel host miRNAs as well as oncogenic compounds that can be repurposed for use in anti-retroviral therapies targeted against HIV-1.